Actinomycetes are branched filamentous Gram-positive bacteria. Streptomyces, Micromonospora, Nocardia, Actinoplanes, Saccharopolyspora, Actinomadura, Thermomonospora, Microbispora, Streptosporangium and others all represent genera of the Actinomycetes (Atlas of Actinomycetes, Asakura Publishing Co., Ltd 1996). Actinomycetes are very important industrially because they produce a variety of secondary metabolites such as antibiotics, herbicides, anticancer agents, antihelmintics, and anabolic agents (Demain., Appl. Microbiol and Biotechnology., 1999, 52:455–463). Antibiotics are a large and complex group of chemical substances which exert deleterious effects on other organisms, many of which organisms are harmful to humans. Thus, antibiotics are particularly important secondary metabolites to study and produce. This is especially true because many pathogens can develop antibiotic resistance to known antibiotics.
Given the actinomycetes' proclivity for producing secondary metabolites such as antibiotics, it is especially advantageous to develop new tools such as vectors, promoters and the like to allow actinomycetes to be easily genetically manipulated. These tools would make it possible to control the levels of expression of genes encoding for secondary metabolites and also would make it possible to prepare derivatives or intermediates of these metabolites. In addition, the development of new vectors utilizing novel genes would make it possible to program microorganisms such as actinomycetes to produce recombinant products such as hybrid antibiotics via genetic engineering techniques.
Integrating vectors are vectors which integrate into a transformed host's chromosome rather than replicating autonomously. They are particularly useful in transforming actinomycetes because they allow for the especially efficient production of secondary metabolites because of their high transformation rates, site specific integrative capacity and stable maintenance in host chromosomes without antibiotic selection.
Vectors have been developed for use in actinomycetes that contain att/int functions for site-specific integration of plasmid DNA. The two systems available make use of the att/int functions of bacteriophage phiC31 (U.S. Pat. No. 5,190,870) and plasmid pSAM2 (U.S. Pat. No. 5,741,675). However, there is a need for additional vectors with att/int functions for site-specific integration in M. carbonacea and similar organisms.
The present inventors have responded to the above needs and have isolated genes from the actinomycete, Micromonospora carbonacea var. africana (ATCC39149, SCC1413) lysogenic phage pMLP1, in order to create vectors which can be used for site-specific integration into Micromonospora chromosomes. These integrating vectors can be used to express actinomycete genes, manipulate secondary metabolic pathways and create new metabolic products such as hybrid antibiotics.